1. Field of the Invention
The field of this invention is enzymes involved in signal transduction.
2. Background
Interleukin 1 (IL-1) receptor associated kinase (IRAK) functions as an intracellular signal transducer for the pro-inflammatory cytokine IL-1. IL-1 treatment of cells induces the complex formation of the two IL-1 receptor chains, IL-1R1 and IL-1RAcP, which recruits an adaptor molecule designated as MyD88 which binds to IRAK. IRAK is subsequently phosphorylated, released from the receptor complex to interact with TRAF6. TRAF6 triggers either the NIK/IKK kinase cascade to activate the transcription factor NF-xcexaB or an undefined kinase cascade to activate the transcription factor AP-1. Both transcription factors regulate large numbers of genes that regulate immune and inflammatory responses.
The genome project has facilitated the identification of a large number of membrane bound receptor-like molecules that are related to IL-1RI and IL-1RAcP by sequence homology. One member of this family, IL-1RrP, has been recently shown to function as a receptor of an IL-1 related cytokine, IL-18, that regulates immune response by promoting the production of interferony. Like IL-1RI and IL-1RAcP, IL-1RrP signals NF-xcexaB activation. Gene disruption experiments and biochemical analyzes indicate that IL-1RrP also utilizes MyD88 and IRAK and TRAF6 as intracellular signal transducers.
Although MyD88 deficient mice failed to respond to IL-1 and IL-18, IRAK deficient mice still have a residual response to IL-1. This observation indicates that other molecules in the cells can partially substitute for the function of IRAK in IL-1 signaling. Recently, an IRAK-related molecule designated IRAK2, was described. Although upon over expression, IRAK2 could interact with IL-1R and IRAK, it has not been shown to be recruited to the receptor complex after IL-1 treatment like IRAK. Therefore, we searched for molecules that can substitute for IRAK in an IL-1 response.
The invention provides methods and compositions relating to isolated IRAK3 polypeptides and related polynucleotides having IRAK3-specific structure and activity. The subject IRAK3 polypeptides and polynucleotides can regulate cellular responsiveness to cytokine activation and hence provide important regulators of cell function. The polypeptides may be produced recombinantly from transformed host cells from the subject IRAK3 polypeptide encoding nucleic acids or purified from mammalian cells. The invention provides isolated IRAK3 hybridization probes and primers capable of specifically hybridizing with the disclosed IRAK3 gene, IRAK3-specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis (e.g. genetic hybridization screens for IRAK3 transcripts), therapy (e.g. IRAK3 kinase inhibitors to inhibit IL-1 induced signal transduction) and in the biopharmaceutical industry (e.g. as immunogens, reagents for isolating other transcriptional regulators, reagents for screening chemical libraries for lead pharmacological agents, etc.).
The nucleotide sequence of a natural human cDNA encoding a human IRAK3 polypeptide is shown as SEQ ID NO: 1, and the full conceptual translate is shown as SEQ ID NO: 2. To clone this novel IRAK3 cDNA, we used a the cDNA insert from a mouse EST clone (AA840598, Genome Systems) as a hybridization probe to screen a lambda cDNA library constructed with Phytohemagglutinin-L (PHA-L) activated human peripheral blood leukocytes under low stringent conditions. We isolated a 2.2 kb cDNA clone with an open reading frame encoding 617 amino acids, and determined that the methionine at position 22 of the open reading frame is the first amino acid of the full-length IRAK3 protein, which consists of 596 amino acids with a calculated molecular weight of 67675 daltons. We determined that IRAK3 can function as a signaling molecule for either IL-1 or cytokines that signal through IL-1R related receptors, can bind MyD88 in coexpression and in vitro binding assays, and plays a role in inflammatory responses and/or immune regulation and thus provides a drug target for treatment of inflammatory diseases and immune disorders.
The IRAK3 polypeptides of the invention include incomplete translates of SEQ ID NO: 1 which translates and fragments of SEQ ID NO: 2 have human IRAK3-specific amino acid sequence, binding specificity or function. Preferred translates/deletion mutants comprise at least 10, preferably at least 15, more preferably at least 25, more preferably at least 35, most preferably at least 50 consecutive residues of SEQ ID NO: 2, preferably of at least one of SEQ ID NO: 2, residues 1-99, residues 100-199, residues 200-299, residues 300-399, residues 400-499 and residues 500-596. The subject domains provide IRAK3 domain specific activity or function, such as IRAK3-specific kinase or kinase inhibitory activity, IRAK-3 specific MyD88-binding or binding inhibitory activity, IRAK3 specific antibody binding or binding inhibitory activity.
IRAK3-specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays: e.g. in vitro binding assays, cell culture assays, in animals (e.g. gene therapy, transgenics, etc.), etc. Binding assays encompass any assay where the molecular interaction of an IRAK3 polypeptide with a binding target is evaluated. The binding target may be a natural intracellular binding target such as an IRAK3 substrate, a IRAK3 regulating protein or other regulator that directly modulates IRAK3 activity or its localization such as MyD88; or non-natural binding target such a specific immune protein such as an antibody, or an IRAK3 specific agent such as those identified in screening assays such as described below. IRAK3-binding specificity may assayed by kinase activity or binding equilibrium constants (usually at least about 107Mxe2x88x921, preferably at least about 108Mxe2x88x921, more preferably at least about 109Mxe2x88x921), by the ability of the subject polypeptide to function as negative mutants in IRAK3-expressing cells, to elicit IRAK3 specific antibody in a heterologous host (e.g a rodent or rabbit), etc. In any event, the IRAK3 binding specificity of the subject IRAK3 polypeptides distinguishes any discernable translation product of EST clone AA840598.
In a particular embodiment, the subject domains provide IRAK3-specific antigens and/or immunogens, especially when coupled to carrier proteins. For example, peptides corresponding to IRAK3-specific domains are covalently coupled to keyhole limpet antigen (KLH) and the conjugate is emulsified in Freunds complete adjuvant. Laboratory rabbits are immunized according to conventional protocol and bled. The presence of IRAK3-specific antibodies is assayed by solid phase immunosorbant assays using immobilized IRAK3 polypeptides of SEQ ID NO: 2, see, e.g. Table 2.
The claimed IRAK3 polypeptides are isolated or pure: an xe2x80x9cisolatedxe2x80x9d polypeptide is unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, and more preferably at least about 5% by weight of the total polypeptide in a given sample and a pure polypeptide constitutes at least about 90%, and preferably at least about 99% by weight of the total polypeptide in a given sample. The IRAK3 polypeptides and polypeptide domains may be synthesized, produced by recombinant technology, or purified from mammalian, preferably human cells. A wide variety of molecular and biochemical methods are available for biochemical synthesis, molecular expression and purification of the subject compositions, see e.g. Molecular Cloning, A Laboratory Manual (Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, N.Y.) or that are otherwise known in the art.
The invention provides binding agents specific to the claimed IRAK3 polypeptides, including substrates, agonists, antagonists, natural intracellular binding targets, etc., methods of identifying and making such agents, and their use in diagnosis, therapy and pharmaceutical development. For example, specific binding agents are useful in a variety of diagnostic and therapeutic applications, especially where disease or disease prognosis is associated with improper utilization of a pathway involving the subject proteins, e.g. NF-xcexaB activation. Novel IRAK3-specific binding agents include IRAK3-specific receptors, such as somatically recombined polypeptide receptors like specific antibodies or T-cell antigen receptors (see, e.g Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory) and other natural intracellular binding agents identified with assays such as one-, two- and three-hybrid screens, non-natural intracellular binding agents identified in screens of chemical libraries such as described below, etc. Agents of particular interest modulate IRAK3 function, e.g. IRAK3-dependent transcriptional activation, esp. dominant negative deletion mutants, etc. Accordingly, the invention also provides methods for modulating signal transduction involving IL-1 receptor activation in a cell comprising the step of modulating IRAK3 kinase activity, e.g. by contacting the cell with a dominant negative IRAK3 deletion mutant, or IRAK3 polynucleotide (below).
The amino acid sequences of the disclosed IRAK3 polypeptides are used to back-translate IRAK3 polypeptide-encoding nucleic acids optimized for selected expression systems (Holler et al. (1993) Gene 136, 323-328; Martin et al. (1995) Gene 154, 150-166) or used to generate degenerate oligonucleotide primers and probes for use in the isolation of natural IRAK3-encoding nucleic acid sequences (xe2x80x9cGCGxe2x80x9d software, Genetics Computer Group, Inc, Madison Wis.). IRAK3-encoding nucleic acids used in IRAK3-expression vectors and incorporated into recombinant host cells, e.g. for expression and screening, transgenic animals, e.g. for functional studies such as the efficacy of candidate drugs for disease associated with IRAK3-modulated cell function, etc.
The invention also provides nucleic acid hybridization probes and replication/amplification primers having a IRAK3 cDNA specific sequence comprising SEQ ID NO: 1 or fragments thereof, and sufficient to effect specific hybridization thereto (i.e. specifically hybridize with SEQ ID NO: 1, particularly in the presence of EST clone AA840598. Such primers or probes are at least 12, preferably at least 24, more preferably at least 36 and most preferably at least 96 bases in length. Demonstrating specific hybridization generally requires stringent conditions, for example, hybridizing in a buffer comprising 30% formamide in 5xc3x97SSPE (0.18M NaCl, 0.01M NaPO4, pH7.7, 0.001M EDTA) buffer at a temperature of 42xc2x0 C. and remaining bound when subject to washing at 42xc2x0 C. with 0.2xc3x97SSPE; preferably hybridizing in a buffer comprising 50% formamide in 5xc3x97SSPE buffer at a temperature of 42xc2x0 C. and remaining bound when subject to washing at 42xc2x0 C. with 0.2xc3x97SSPE buffer at 42xc2x0 C. IRAK3 nucleic acids can also be distinguished using alignment algorithms, such as BLASTX (Altschul et al. (1990) Basic Local Alignment Search Tool, J Mol Biol 215, 403-410).
The subject nucleic acids are of synthetic/non-natural sequences and/or are isolated, i.e. unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, preferably at least about 5% by weight of total nucleic acid present in a given fraction, and usually recombinant, meaning they comprise a non-natural sequence or a natural sequence joined to nucleotide(s) other than that which it is joined to on a natural chromosome. Recombinant nucleic acids comprising the nucleotide sequence of SEQ ID NO: 1, or fragments thereof, contain such sequence or fragment at a terminus, immediately flanked by (i.e. contiguous with) a sequence other than that which it is joined to on a natural chromosome, or flanked by a native flanking region fewer than 10 kb, preferably fewer than 2 kb, which is at a terminus or is immediately flanked by a sequence other than that which it is joined to on a natural chromosome. While the nucleic acids are usually RNA or DNA, it is often advantageous to use nucleic acids comprising other bases or nucleotide analogs to provide modified stability, etc.
The subject nucleic acids find a wide variety of applications including use as translatable transcripts, hybridization probes, PCR primers, diagnostic nucleic acids, etc.; use in detecting the presence of IRAK3 genes and gene transcripts and in detecting or amplifying nucleic acids encoding additional IRAK3 homologs and structural analogs. In diagnosis, IRAK3 hybridization probes find use in identifying wild-type and mutant IRAK3 alleles in clinical and laboratory samples. Mutant alleles are used to generate allele-specific oligonucleotide (ASO) probes for high-throughput clinical diagnoses. In therapy, therapeutic IRAK3 nucleic acids are used to modulate cellular expression or intracellular concentration or availability of active IRAK3.
The invention provides efficient methods of identifying agents, compounds or lead compounds for agents active at the level of a IRAK3 modulatable cellular function. Generally, these screening methods involve assaying for compounds which modulate IRAK3 interaction with a natural IRAK3 binding target such as MyD88. A wide variety of assays for binding agents are provided including labeled in vitro proteinxe2x80x94protein binding assays, immunoassays, cell based assays, etc. The methods are amenable to automated, cost-effective high throughput screening of chemical libraries for lead compounds.
Identified reagents find use in the pharmaceutical industries for animal and human trials; for example, the reagents may be derivatized and rescreened in in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development.
In vitro binding assays employ a mixture of components including an IRAK3 polypeptide, which may be part of a fusion product with another peptide or polypeptide, e.g. a tag for detection or anchoring, etc. The assay mixtures comprise a natural intracellular IRAK3 binding target. In a particular embodiment, the binding target is an MyD88-derived binding peptide or an IRAK3-derived substrate of IRAK3 kinase activity. While native full-length binding targets may be used, it is frequently preferred to use portions (e.g. peptides) thereof so long as the portion provides binding affinity and avidity to the subject IRAK3 polypeptide conveniently measurable in the assay. The assay mixture also comprises a candidate pharmacological agent. Candidate agents encompass numerous chemical classes, though typically they are organic compounds; preferably small organic compounds and are obtained from a wide variety of sources including libraries of synthetic or natural compounds. A variety of other reagents may also be included in the mixture. These include reagents like ATP or ATP analogs (for kinase assays), salts, buffers, neutral proteins, e.g. albumin, detergents, protease inhibitors, nuclease inhibitors, antimicrobial agents, etc. may be used.
The resultant mixture is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, the IRAK3 polypeptide specifically binds the cellular binding target, portion or analog with a reference binding affinity. The mixture components can be added in any order that provides for the requisite bindings and incubations may be performed at any temperature which facilitates optimal binding. Incubation periods are likewise selected for optimal binding but also minimized to facilitate rapid, high-throughput screening.
After incubation, the agent-biased binding between the IRAK3 polypeptide and one or more binding targets is detected by any convenient way. For IRAK3 kinase assays, xe2x80x98bindingxe2x80x99 is generally detected by a change in the phosphorylation of an IRAK3 substrate. In this embodiment, kinase activity may quantified by the transfer to the substrate of a labeled phosphate, where the label may provide for direct detection as radioactivity, luminescence, optical or electron density, etc. or indirect detection such as an epitope tag, etc. A variety of methods may be used to detect the label depending on the nature of the label and other assay components, e.g. through optical or electron density, radiative emissions, nonradiative energy transfers, etc. or indirectly detected with antibody conjugates, etc.
A difference in the binding affinity of the IRAK3 polypeptide to the target in the absence of the agent as compared with the binding affinity in the presence of the agent indicates that the agent modulates the binding of the IRAK3 polypeptide to the IRAK3 binding target. Analogously, in the cell-based assay also described below, a difference in IRAK3-dependent transcriptional activation in the presence and absence of an agent indicates the agent modulates IRAK3 function. A difference, as used herein, is statistically significant and preferably represents at least a 50%, more preferably at least a 90% difference.
The following experimental section and examples are offered by way of illustration and not by way of limitation.